The invention concerns a determination method and equipment as well as an adapter for use in these. The invention is especially applicable to automatic immunodetermination systems.
Solid-phase immunodetermination is usually performed in one vessel so that the analyte to be determined and possibly contained in the sample is first allowed to react with a separating reagent bound in a solid phase, whereupon the other steps required in the determination are performed in the same vessel. The troublesome thing here is that much dosing and removing of liquids must be performed. When several different determinations are done, a large stock of different reagents is also needed.
A system is also known wherein the solution to be used in each determination step is placed in advance in its own vessel. The solid phase is formed by the inside surface of a disposable pipette jet. In each step the pipette jet is brought into the respective vessel, the solution is drawn into the jet and a reaction is allowed to take place, whereafter the jet is emptied and moved into the next vessel. During the step the solution is moved back and forth in the jet. The equipment has several suction cylinders with pumps so that several determinations can be performed in parallel. No exact dosing devices are required in this equipment. Nor are any reagent containers required in the equipment. However, the drawback is that through a vapour phase samples are in connection with the cylinders of the equipment which can not, however, be washed automatically. This can cause a risk of contamination. Liquid will also remain in the pipette jet and will move along to the following step. In addition, piston pumps wear easily and unpredictably, for which reason their condition must be checked often. Another problem is the sealing of the pipette jet to the suction cylinder. All things considered, much trouble can occur in this device. Besides, there is only limited solid-phase surface area available on the inner surface of the pipette jet.
A method of determination as defined in claim 1 has now been invented. Advantageous applications of the same are presented in the other claims.
As used herein, a separating reagent means such a substance which reacts with the analyte to be determined and binds it in a solid phase. In immunodeterminations the separating reagent is usually an antigen or an antibody. A medium here generally means a solution, such as a reaction solution or a washing fluid, to be used in some determination step.
The outer surface of solid particles separate from the reaction vessel is used as the solid phase in the method and the determination steps are carried out in two or several vessels. The particles are moved from one vessel to another using a special remover. The particles are kept in the vessel containing the sample and a separating reaction is allowed to take place. Then any other required steps are performed in other vessels, and finally the particles are moved to the measuring vessel. Mediums needed for the determination are dosed beforehand into the vessels.
The particles are preferably magnetic particles, whereby the remover preferably contains a magnet which can be moved in relation to the remover.
The vessels are preferably formed as one unit. In principle, however, some steps, especially measuring of the formed reaction product, can be performed outside the vessel unit, if desired. An outside measuring vessel could be used especially when the complex is detected directly from the solid phase, for example, fluorometrically or radiometrically.
Correspondingly, several steps, e.g. washes, can also be performed in the same vessel. A medium can also be dosed into some vessel or removed from it. Separate dosings could possibly be used in those steps where exact dosing is not necessary and where, for example, the same medium is used in several different determinations. Washes, in particular, could be such steps. However, normally such vessel units are more advantageous where all different mediums are ready in different vessels.
At least washes are usually performed in intermediate determination steps. In addition, the resulting reaction complex is usually joined in a middle step to a tracer which is then detected in the measuring step. The tracer can be either directly detectable or it can be a tracer which releases a detectable compound from a special substrate. Detection usually takes place fluorometrically, luminometrically, absorptiometrically or radiometically.
There is no risk of contamination in the method, because the sample is not drawn into the equipment from the plate vessels. In addition, the method can be carried out using simple and very reliably-operating automatic equipment.
The invention is suitable, for example, for immunologic, DNA-hybridization or hormone determinations.
The remover surface is preferably such that liquid will run off it as completely as possible. Preferably there is also a tip at the bottom end. The bottom of the reaction vessel is advantageously designed with the same shape as the remover, whereby as little medium as possible will be needed.
A very large solid-phase surface area is obtained by using solid-phase particles which are separate from the remover. The most advantageous ones are so-called microparticles. Magnetic particles are preferably used which are made to adhere easily to the remover with the aid of a magnet.
When using non-magnetic separate particles, the remover is provided, for example, with a grid or a filter to separate the particles from the medium.
To speed up mass transfer and thus also the necessary reaction time, the medium is preferably agitated during the reaction. This is preferably done by moving the remover. It is especially advantageous to move the remover in a vertical direction, whereby the medium must flow through a gap between the vessel and the remover, thus blending very effectively. To make blending more effective the remover is made so wide that a gap of a suitable narrowness is formed between the vessel and the remover. Agitation can also be promoted by a suitable remover and vessel design.
The vessel unit forms a plate for use in one determination. The remover can be packed into some vessel in the plate. The vessels for use in different steps may also be of different sizes.
The vessels are preferably closed with a film, which is punctured while carrying out the method. The film can be punctured by using the remover, but a separate puncturing point may also be used. The point may have cutting blades which form strips which tear in a controlled manner. The puncturing point may be attached to the same actuator as the remover in the equipment. The top edge of the vessel preferably has an extension against which the strips of the punctured film can rest. Closed vessels may contain an inert vapour phase to improve durability.
The equipment can also have a safeguarding system, which will make sure before the step is started that the vessel contains a medium. The remover may work conveniently as the indicator of such a system based on electric conductivity measurement.
If desired, in that reaction vessel in particular into which the sample is brought some suitable substance may be fastened to the vessel wall or to a separate solid phase remaining in the vessel, which substance binds such substances from the sample or from the formed complex which may disturb later determination steps.
The plate vessels are preferably in a single straight row, whereby the remover need be moved only along a straight path in the horizontal plane in relation to the plate. The vessels for the different steps may be located in any order in relation to each other. The vessels are preferably permanently fixed to one another. The plate may be made of some suitable material, preferably of plastic.
The plate is advantageously provided with detents and the equipment provided with their counterparts, so that the plate can not be located in a wrong position by mistake.